Late stage assay provider counterscreen results from the probe development effort to identify non-agonists of the peroxisome proliferator-activated receptor gamma (PPARg): chemiluminescence-based biochemical western blot assay for inhibitors of phosphorylation of PPARG protein levels

Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor superfamily and are lipid sensors functioning as ligand-dependent transcription factors regulating gene expression patterns of diverse biological processes (1, 2). PPARs play a critical role in metabolic processes such as glucose metabolism, lipid metabolism, and have been implicated in anti-atherogenic, anti-inflammatory as well as anti-hypertensive functions (3). Like other nuclear receptors, PPARs act as agonist-activated transcription factors, regulating specific PPARG gene transcription. PPARs have been shown to respond to small molecules and are well-documented for therapeutic actions triggered by synthetic agonists (4-6). Among the three isoforms of PPAR identified, PPAR gamma (NR1C3) is implicated in several important disorders such as atherosclerosis, diabetes, obesity and cancer, providing strong justification for the search for specific PPARg agonists that can be used to treat these pathologies. However, the clinical use of PPARg agonists has been associated with adverse effects that are mainly caused by the concomitant activation of various PPARG genes implicated in different physiological pathways. These side effects include weight gain through increased adipogenesis, renal fluid retention and plasma volume expansion, as well as toxic effects in the liver (7). To design safer and more selective PPARg agonists, the different physiological pathways triggered by PPARg activation have to be decoupled. Recently, new classes of PPARg ligands, the so called selective PPARg modulators (SPPARgMs), have been developed. These compounds respond as partial agonists in a GAL-4 luciferase assay and are assumed to display a different binding mode in the PPARg subunit compared to the full agonist, glitazones (8). Selective recruitment of transcriptional coactivators by partial agonists has also been demonstrated, suggesting that different PPARg binding mode leading to a distinct coactivator recruitment profile may explain the change in gene expression patterns compared to those of full agonists (glitazones). Further, due to their improved pharmacodynamic properties, there is substantial interest and need to develop insulin-sensitizing PPARg modulators with minimal classical activation of PPARg and reduced side effects, while maintaining robust antidiabetic efficacy (9-11). The objective of this project is to identify compounds that bind to PPARgamma and do not induce PPARg transactivation (ligands; non-agonists) (12).

Assay Overview:The purpose of this assay is to determine whether powder samples of a compound identified as a novel PPARG ligand and non-agonist probe candidate (ML244) modulates the expression of PPARg gene involved in the differentiation of adipocytes. The probe should not be active in this assay. This assay employs the differentiated mouse 3T3-L1 cell line. The compound was tested at 10 uM.Protocol Summary:3T3-L1 cells were cultured in Alpha-MEM supplemented with 10% FBS, 1% penicillin-streptomycin, 200 uM ascorbic acid and 10 mM-glycerophosphate. The cells were treated with either Rosiglitazone (10 uM) or SR1664 (10 uM) or left in vehicle at the start of differentiation. The cells were harvested 7 days post-differentiation for gene expression analysis and 21 days post-differentiation for mineralization. The mineralization of MC3T3-E1 cells was determined by Alizarin red S staining (Millipore ECM815) as per manufacturer's instructions. Total RNA was isolated from cells using Trizol reagents. The RNA was reverse-transcribed using the ABI reverse transcription kit. Quantitative PCR reactions were performed with SYBR green fluorescent dye using an ABI9300 PCR machine. Relative mRNA expression was determined by the delta-delta-Ct method. The gene expression was normalized to GAPDH and compared to levels in cells treated with DMSO (mRNA level set as 1.0).PubChem Activity Outcome and Score:Compounds that modified (increased or decreased) gene expression by 200% or greater (ie, induced a 2X-fold change in expression) compared to the DMSO levels (set at 1.0) were active in this assay. Compounds that modified (increased or decreased) gene expression to levels less than 200% of the DMSO level (set as 1.0) were active in this assay. The probe was inactive.The PubChem Activity Score is assigned a value of 100 for probe compounds, 50 for actives and 0 for inactives.The PubChem Activity Score range for inactive compounds is 0-0. There are no active compounds.List of Reagents:Differentiated 3T3-L1 cells; Trizol Reagent (Invitrogen)ABI Reverse Transcription Kit; SYBR Green dye (ABI)PCR primers were ordered as appropriate. The primer pair sequences for PPARg gene target is indicated below:Forward: GCATGGTGCCTTCGCTGAReverse: TGGCATCTCTGTGTCAACCATG

This assay was run by the assay provider. This assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. Possible artifacts of this assay can include, but are not limited to cytotoxic compounds and compounds that modulate protein synthesis or processing. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided.